FIGURE 4.

TLP-responsive element within human TAp63 promoter. A, introduction of the mutations into the putative TLP-responsive element. We have introduced the mutations (5′-CTAGTGAGCA-3′) into the putative TLP-responsive element (5′-AGCTGGAGCA-3′) within pGL3-TAp63(−2340) to give pGL3-TAp63(−2340/M1). HeLa cells were transiently co-transfected with the constant amount of Renilla luciferase reporter plasmid together with pGL3-TAp63(−2340) or with pGL3-TAp63(−2340) in the presence of the expression plasmid for FLAG-TLP (closed bars) or empty plasmid (open bars). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. B, functional significance of the putative TLP-responsive element. We have generated the luciferase reporter construct bearing four tandem repeats of the putative TLP-responsive element (5′-AGCTGGAGCA-3′) fused to just upstream of TAp63 core promoter termed pGL3-TAp63-TLP-luc and its mutant carrying four tandem repeats of mutant form of the canonical TLP-responsive element (5′-CTAGTGAGCA-3′) fused to just upstream of TAp63 core promoter termed pGL3-TAp63-M1-luc. HeLa cells were transiently co-transfected with the constant amount of Renilla luciferase reporter plasmid along with the constant amount of pGL3-TAp63-TLP-luc or with pGL3-TAp63-M1-luc in the presence of FLAG-TLP expression plasmid or the empty plasmid. Eighteen hours after transfection, cells were lysed, and their luciferase activities were determined. Open and closed bars indicate the relative luciferase activity in cells transfected with the empty plasmid and the expression plasmid for FLAG-TLP, respectively. N.S., not significant.